Summary

Glutamate 5-kinase (G5K) catalyzes the first step in the L-proline biosynthesis pathway. G5K activity is feedback-inhibited by L-proline, providing the primary point of control in this pathway [Hayzer80, Smith84, PerezArellano05, PerezArellano06]. Although G5K activity produces the highly reactive intermediate γ-L-glutamyl 5-phosphate (GP), suggesting channeling of this compound to the following enzyme, a stable complex between G5K and glutamate-5-semialdehyde dehydrogenase could not be isolated [Smith84]. The channeling of GP has subsequently been challenged by evidence that a protein-protein interaction is not required between G5K and ProA for proline synthesis, and the measured half-life of GP is long enough to reach ProA [Newton24].

It has been observed that in a proB mutant, which is blocked in L-proline biosynthesis, many single mutations occurred in glnA that resulted in leaking of γ-L-glutamyl 5-phosphate into the intracellular milieu, enabling proline biosynthesis [MedinaCarmona23].

G5K consists of an N-terminal amino acid kinase (AAK) domain and a C-terminal PUA (PseudoUridine synthases and Archaeosine-specific transglycosylases) domain. Deletion of the PUA domain of G5K has demonstrated that the AAK domain alone, like the full-length protein, can form tetramers, catalyze the formation of L-glutamate-5-phosphate and is feedback inhibited by proline. However, the deletion greatly diminished the Mg²⁺ requirement of the enzyme and reduced its sensitivity to proline-mediated feedback inhibition [PerezArellano05]. Site-directed mutagenesis has confirmed that the AAK domain is involved in substrate binding, catalysis, and feedback inhibition by proline [PerezArellano06].

Crystal structures of G5K have been solved [PerezArellano04, MarcoMarin07]. G5K possesses a "dimer of dimers" architecture in which the tetramers are formed by the interaction of two dimers through their AAK domains [MarcoMarin07]. While the enzyme purifies as a tetramer, the functional unit appears to be the dimer [PerezArellano10]. The binding sites for glutamate (substrate) and proline (inhibitor) overlap [PerezArellano06, MarcoMarin07, PerezArellano10]. A flexible loop in the active site cavity modulates the interaction of both proline and glutamate with G5K [PerezArellano10].

Overproduction of proline is osmoprotective; mutations in proB that enhance osmotic stress tolerance are causing a loss of feedback inhibition of G5K by proline [Mahan83, Smith85, Csonka88].

proAB is part of a genomic region that is commonly deleted in E. coli clones isolated from long-term stab cultures [Faure04]. Suppressor mutations that allow a ΔFRAME: EG10418|| strain to grow in the presence of arsenate map to proA and proB, enabling synthesis of γ-glutamyl cysteine, which is converted to glutathione by GshB. The suppressor alleles of G5K reduce the K_m for glutamate and relieve proline-mediated feedback inhibition in the presence of a proA null allele [Veeravalli11].

ProB may have a physiologically relevant RNA binding activity [Klein23].

Review: [Csonka07]

Additional Citations: [Baich65, Broda74, Berg74, Inuzuka76, Inuzuka76a, Hayzer78, Hayzer78a, Shaw79, Hayzer83a, Deutch84, Rushlow85, Garcia85, Nersisian86, Seddon89, Neumyvakin90, Chattopadhyay04, Joyce06, Blank14]